Guidelines (Following is our recommended protocol. This protocol only provides a guideline, and should be modified according to your specific needs).
Detection of infiltrating Mast cells^[1]:
1. Tissue processing
1) Immersed tissue samples in 10% buffered formalin for24 h.
2) Remove tissues from the fixative and place in 70% alcohol.
3) Embed in paraffin: sequentially incubate in 70, 85, 95, and 100% ethanol, xylene, and molten paraffin (30 minincubation each), and set tissues in paraffin blocks.
4) Cut paraffin-embedded tissue in 5-7 μm sections and place onto glass slides.
5) Bake the slides for a minimum of1 hat 55°C before use.
2. Tissue staining
1) De-wax paraffin-embedded tissue sections by immersing slides5 minin xylene (or Histo-Clear), and rehydrate tissue by sequential immersions (5 mineach) in 100%, 95%, and 70% ethanol and a final2 minimmersion in distilled water.
2) Place the slides in Harris' hematoxylin solution for70 s.
3) Briefly wash the excess of hematoxylin by immersing the slides two or three times in clean tap water. Briefly tap the slides on paper towels to remove excess water.
4) Immerse slides briefly three times in 70% alcohol, and wash again in clean tap water for3 min. Remove excess water.
5) Place slides in Scott's bluing solution for up to a minute. This step gently transforms hematoxylin into an insoluble blue color within the nucleus.
6) Inspect the slides under the bright-light microscope to ensure the nuclei are properly stained.
7) Wash slides in clean tap water for3 min, and remove excess water.
8) Immerse slides in clean~1.1%Toluidine Blue purity 36% solution for4 min(Note 1).
9) Wash slides in clean tap water for3 minand remove excess water.
10) Quickly dip the slides three times in one batch of 70% ethanol and then dip them five times in 5% eosin solution.
11) Dehydrate the slides by successive immersion in 70% (2 min), 95% (twice, 2 mineach), and 100% ethanol (twice, 2 mineach); conclude this step by immersing the slides in xylene (or Histo-Clear) (twice, 3 mineach).
12) Permanently mount each slide with a clean coverslip using xylene-based mounting medium.
13) Let slides dry for a minimum of60 minbefore analyzing them under the bright-light microscope.
Note 1: 1.1% Toluidine Blue purity 36% solution: mix1.1 gToluidine Blue purity 36% with100 mL0.1 Msodium acetate buffer pH 4. Stir well. Adjust the pH to 2.0-2.5 by adding drops of1 Mhydrochloric acid. Staining is performed at 25 ℃, protected from light. Ready to use.
Application of Toluidine Blue purity 36%^[2][3]:
1. Connective tissue mucins, especially acid mucins. The tissue stains purple to red, while the background is stained blue.
2. Mast cell granules stain purple in color due to the presence of heparin and histamine.
3. Amyloid stain blue but under polarized light they give a bright red birefringence.
4. Endocrine cell granules are stained purple to red (concentration of stain is0.01%).
5. Sulfatides stain red brown or yellow. Only lipids that are sufficiently acidic to induce a metachromatic shift are stained.
6. Corneybacterium diphtheria contains granules with polymerized inorganic polyphosphate, which stains red violet color.
7.Helicobacterstains dark blue against a variably blue background (concentration of stain is1%).
8. Toluidine Blue purity 36% can be used to stain frozen section because of the rapidity of the staining procedure (10-20 s) and better clarity of the cells.9. Toluidine Blue purity 36% can also dye the lignins of the plant blue-green, the phloem blue-purple, and the rest of the plant pale blue-green.